The sample holder is a component of an electron microscope providing the physical support for samples under observation. To use the sample holder, one or more samples are first placed on a sample support device. The sample support device is then mechanically fixed in place at the specimen tip, and the sample holder is inserted into the electron microscope through a load-lock. During insertion, the sample holder is pushed into the electron microscope until it stops, which results in the specimen tip of the sample holder being located in the column of the microscope. To maintain an ultra-high vacuum environment inside the electron microscope, flexible o-rings are typically found along the barrel of the sample holder, and these o-rings seal against the microscope when the sample holder is inserted.
Certain sample holders can be used to provide a means for gas or liquid to flow into and out of a cavity at the tip of the holder (see, for example FIGS. 1 and 2). These sample holders include devices, e.g., semiconductor devices, which are designed with relatively thin electron beam transparent membranes, positioned in the cavity at the tip of the holder. To establish temporary or continuous flow of liquid or gas, a pump located external to the sample holder can be used to force liquids into the cavity at the tip of the holder, including between two MEMS devices which define an environmental cell. Since the pumping equipment is outside of the holder, various connectors are used to bring the liquid to the sample holder, down the length of the holder, to the cavity at the tip of the holder, and back out of the sample holder. Use of a pump to flow the liquid is typical, but any method of creating a pressure differential could be used to establish flow. For example, a pressurized reservoir on the entry port or a depressurized reservoir on the exit port(s) would also establish flow.
One type of sample support device is an environmental cell which comprises two semiconductor devices, i.e., MEMS devices, comprising thin membrane windows and samples positioned between the semiconductor devices, wherein the sample's environment, including an electrical field and a gas or liquid flow, can be precisely controlled. The present inventors previously described novel apparatuses and methods to contact and align devices used to form liquid or gas cells in International Patent Application No. PCT/US2011/46282 filed on Aug. 2, 2011 entitled “ELECTRON MICROSCOPE SAMPLE HOLDER FOR FORMING A GAS OR LIQUID CELL WITH TWO SEMICONDUCTOR DEVICES,” which is hereby incorporated herein in its entirety.
There are many reasons why environmental cell users desire liquid to flow either intermittently or continuously: flow provides a means to keep the sample hydrated; flow allows the user to create a reaction that can be viewed in the microscope real time; and a system that includes at least three ports allows users to combine two or more fluids within the cavity at the tip of the holder.
The environmental cells are typically designed such that the two semiconductor devices are substantially parallel to one another and positioned about 50 nm to about 5 μm relative to one another. This ensures small liquid layers therebetween, which maximizes the microscope resolution of the sample, which becomes less resolute as the electron beam of the microscope travels through greater thicknesses of liquid. That said, the typical design of the environmental cells allow much greater volumes of fluid to flow around the semiconductor devices than across them. For example, in the case of a 150 nm environmental cell thickness on a Protochips Poseidon 200 holder, there is approximately 500 times more cross sectional area around the E-chip than across the membrane. This creates difficulties for the users of environmental flow cells:
1.) The electron beam can create heat that can evaporate the liquid in the cell. In many cases, greater flow across the semiconductor devices is needed to replace the volume of gas created by electron beam heating. Increasing the flow rate into the tip of the cell can help, but it brings higher risk of over pressurizing the cavity, potentially causing damage;
2.) Sometimes it is difficult to prepare and/or maintain the desired surface energy of the semiconductor devices. For example, if a surface is hydrophobic, it can be difficult to establish the fluid environment desired for a given experiment.
3.) Flow rates are typically adjusted by the user with an external pump system to attain the desired flow rate for sample imaging. If the majority of liquid flows around the sample area than across it, the flow rates may need to be as high as 150 microliters per hour or even higher. With a design where there is less fluid bypassing the membranes, the flow rate can be decreased. This reduction in flow rate improves safety of the microscope, e.g., in the event of a membrane break, less fluid will be able to escape into the column of the microscope.
4.) Users that want to combine known quantities of two liquids between the semiconductor devices are not able to quantify the ratio of the two fluids at the viewing area, i.e., the membranes of the semiconductor devices. This is because it is not possible to know how much liquid of one fluid bypasses the semiconductor devices as compared to the second fluid. This is due to asymmetry in the tip of the sample holder during assembly;
5.) In some cases, the research benefits from knowing the actual rate of fluid flow. This is especially important for those studying reactions; and
6.) Electrochemistry reactions can require rapid replenishment of the electrolyte liquid to prevent the membrane area from becoming dry.
Accordingly, a fluidic cell that can overcome evaporation effects and provide a known flow volume at of fluid at safe pressures across the sample is needed. Towards that end, an invention is disclosed herein to deliver quantifiable amounts of liquid to the membrane of an environmental holder.